Antitumor hybrid and process for the preparation thereof

ABSTRACT

Antitumor protein hybrid, composed of a moiety which is substantially the fragment Fab of an antitumor immunogloblin and a moiety which is the subunit A of ricin, which is expressed by the following formula (I): 
     
         Fab-S.sub.1 --(X).sub.n --S.sub.2 --RA).sub.m . . .        (I) 
    
     (where Fab indicates a moiety which is substantially the fragment Fab of an antitumor immunogloblin; RA indicates a moiety which is the subunit A of ricin; X indicates a divalent organic radical; S 1  and S 2  are both sulfur atoms, S 1  indicating a sulfur atom arising from the disulfide bond (--S--S-- bond) in an immunoglobulin and S 2  a sulfur atom arising from the disulfide bond in ricin; n stands for 0 or 1 and m stands for an integer of 1 to 5). This antitumor protein hybrid has remarkable and specific cytotoxicity against tumor cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 170,332, filedJuly 21, 1980, now U.S. Pat. No. 4,350,626.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel antitumor protein hybrid, forexample, cytotoxic against mouse tumors, and a process for thepreparation thereof. More particularly, the present invention relates toa novel protein hybrid, which, having a moeity which is substantiallythe fragment Fab of an antitumor immunoglobulin and a moiety which isthe subunit A of plant toxin ricin, is specifically useful as a remedyfor malignant tumor e.g., in mice, and a process for the preparation ofthe same.

2. Description of the Prior Art

As for the remedies for malignant tumor or cancer (antitumor agents)many drugs have hitherto been known; however, these drugs have adisadvantage that they can not be administered enough to destroy tumorcells, because they have a toxic effect not only upon tumor cells butalso upon normal cells to a considerable degree. Several attempts havebeen made to overcome this disadvantage by combining an antitumor agentor a protein toxin having cytotoxicity with a specific carrier in orderto have them selectively absorbed by tumor cells. There exists anantitumor antibody (antitumor immunoglobulin), though very small inamount, in the blood of a cancer patient or on the surface of tumorcells. An antitumor antibody can also be obtained by immunizing ananimal with the tumor cells and absorbing the obtained antiserum withthe human normal cells. Antitumor antibodies, whether autochthonus,allogeneic, or xenogeneic, are not always capable of displaying acytotoxic effect against tumor cells; however, they have a common natureof combining with tumor cells with an extremely high selectivity.Antitumor antibodies, therefore, have been used as a carrier to have anantitumor agent or a protein toxin absorbed by tumor cells selectively.

For instance, Japanese Patent Application Laid-open No. 144723/76discloses, as an antitumor drug, a conjugate of antibody and antitumordrug in which such antitumor drug as daunomycin, etc. is boundcovalently with Fab' dimer of antitumor immunoglobulin. This conjugateis superior in that it carries the antitumor drug selectively to thetarget tumor cells; however, since an antitumor drug itself such asdaunomycin, etc. bound with the antibody (Fab' dimer) still exertscytotoxic effects not only against tumor cells but also against normalcells, it is not satisfactory in view of destroying tumor cells only,and its cytotoxicity itself is not always sufficient either.

Studies have also been made to use diphtheria toxin, which is one of theprotein toxins having much stronger toxicity, in the place of anantitumor drug.

For instance, F. L. Moolten et al. report that they prepared a conjugateby conjugating rabbit anti-SV40 antibody to a diphtheria toxin withglutaraldehyde as a coupling agent and were able to protect hamsterschallenged with SV40-transformed 3T3 cells from developing tumors byadministering the conjugate to hamsters (Journal of the National CancerInstitute, Vol. 55, pp. 473-477, 1975).

P. E. Thorpe et al. report that the conjugate prepared by couplingdiphtheria toxin to antilymphocytic antibody by means of chlorambucilgreatly reduced the protein synthesis of human lymphoblastoid cells,CLA4. (Nature, vol. 271, pp. 752-754, 1978). The results of thesestudies show that a conjugate of diphtheria toxin and antibody displaystoxicity against the tumor cells selectively. However, these conjugates,when used as an antitumor drug, are believed to have some disadvantagesas cited below. First, xenogenic antibody (immunoglobulin) has a strongantigenicity in the human body and induces the formation ofanti-xenogeneic immunoglobulic antibody which deactivates the antitumoractivity and further causes an anaphylaxis shock. The second of thedisadvantages is that the nonspecific toxicity of diphtheria toxin isnot nullified. More particularly, the object of these methods is toconjugate diphtheria toxin on the surface of tumor cells by the aid ofantitumor antibody; however, since the conjugate contains the wholemolecule of diphtheria toxin in its composition, it tends to bind withnormal cell surface receptors for diphtheria toxin and displaycytotoxicity against the normal cells. Thirdly comes the disadvantagewhich is found in the method of cross-linking the antibody with thediphtheria toxin. Many of the cross-linking agents such asglutaraldehyde, toluene diisocyanate, diethyl malonimidate,chlorambucil, etc. effect cross-linking not only between the antibodyand the toxin but also between antibody and antibody, and toxin andtoxin, and moreover, they effect the formation of intramolecule bonds inthe antibody and in the toxin molecule, thus causing the formation ofundesirable products and decrease or loss of the antitumor activity.

SUMMARY OF THE INVENTION

The present inventors have achieved this invention as a result of theirearnest research work to overcome such disadvantages as found with theprior art by developing an antitumor substance which displays strongcytotoxicity against, for example, mice tumor cells selectively.

The present invention relates to an antitumor protein hybrid, composedof a moiety which is substantially the fragment Fab of an antitumorimmunoglobulin and a moiety which is the subunit A of ricin, which isexpressed by the following formula (I):

    Fab--S.sub.1 --(X).sub.n --S.sub.2 --RA).sub.m             (I)

(where Fab indicates a moiety which is substantially the fragment Fab ofan antitumor immunoglobulin; RA indicates a moiety which is the subunitA of ricin; X indicates a divalent organic radical; S₁ and S₂ are bothsulfur atoms, S₁ indicating a sulfur atom arising from the --S--S-- bondin an immunoglobulin and S₂ a sulfur atom arising from the --S--S-- bondin ricin; n stands for 0 or 1 and m stands for a integer of 1 to 5), anda process for preparing said antitumor protein hybrid, which processcomprises binding the sulfur atom in said fragment Fab with the sulfuratom in said subunit A directly or indirectly.

BRIEF DESCRIPTION OF THE DRAWING

In FIG. 1 is a pictorial drawing of a type specimen to show a basicstructure of the immunoglobulin and

FIG. 2 is a pictorial drawing of a type specimen to show a structure ofhuman immunoglobulin IgGl.

In FIG. 3, (a) is a pictorial drawing of a type specimen to show astructure of ricin and (b) is those of subunits A and B.

FIG. 4 shows patterns of electrophoresis conducted with sodium dodecylsulfate-polyacrylamide gel. Disc 1 shows F(ab')₂, disc 2 Fab', disc 3subunit A, disc 4 a polymer of subunit A, and disc 5 a reaction mixturesolution of Fab' and subunit A (Example 1) respectively. Also, disc 6shows peak I of FIG. 4, disc 7 peak II of FIG. 5, disc 8 peak III ofFIG. 5, disc 9 a mixture of F(ab')₂ and peak I, disc 10 a mixture of apolymer of subunit A and peak I, and disc 11 a product obtained bymoderately reducing the protein of peak I with 2-mercaptoethanolrespectively.

FIG. 5 shows the elution pattern obtained by Sephadex G150 columnchromatography conducted for the reaction mixture of fragment Fab' ofanti-L1210 immunoglobulin and subunit A of ricin, and the protein hybridof the present invention is included in the shaded part.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

What is called antitumor immunoglobulin in the present invention is aprotein (immunoglobulin) which has an antibody activity prepared from,forinstance, the serum of a patient with cancer or serum obtained fromsuch animals as monkeys, horses, cows, goats, sheep, rabbits, etc. whichare hyperimmunized with cancer cells or cancer antigen according to apubliclyknown method such as the Cohn ethanol fractionation method,ammonium sulfate fractionation method, ion-exchange chromatography, etc.Or it is aprotein having an antibody activity of high selectively tocancer antigen obtained from a culture fluid of hybridomas or from aserum or ascites of animals inoculated with hybridomas which areprepared by fusing antibody-producing lymphocytes obtained from ananimal immunized with cancer cells or cancer antigen to fuse, forinstance, with myeloma cells (See, for instance, H. Koprowski, et al.,Proc. Natl. Acad. Sci. U.S.A., Vo. 75, No. 7, pp 3405-3409, 1978;Herlyn, et al., ibid., Vol. 76, pp 1438-1442, 1979; M.-Y. Yeh, et al.,Proc. Natl. Acad, Sci. U.S.A., Vol. 76, No. 6, pp 2927-2931, 1979; R. H.Kennett, et al., Science, Vol. 203, pp 1120-1121, 1979.) A protein,which has antibody activity, prepared by isolating an antitumor antibodyfrom a tumor tissue with a denaturant suchas a surface active agent,etc., followed by the same processing procedure as mentioned above, isalso included under the antitumor immunoglobulin according to thepresent invention.

It is known that there are five major classes of immunoglobulins, IgG,IgA,IgM, IgD and IgE, and that their basic structure comprises, as shownby a pictorial drawing of a type specimen in FIG. 1, two L chains whichare indicated by L in the figure and two H chains indicated likewise byH, allchains being bound with at least three disulfide bonds (--S--S--bonds). Toexplain the basic structure of the immunoglobulin shown inFIG. 1, it consists of Fab parts which are shown by Fab in the figureand an Fc part shown by Fc; Fab parts have an antibody activity (what iscalled antitumoractivity in the present invention), or more particularlyan ability to selectively couple to the antigen; Fc part has an abilityto couple to complements or Fc receptors on the cell membrane.

The moiety substantially comprising the fragment Fab which is one moietyofthe antitumor protein hybrid of the present invention corresponds tothe moiety comprising the fragment having an antibody activity arisingfrom said Fab part of the immunoglobulin. For instance, it is known thatIgGl, which is typical of human immunoglobulins, has a structure shownby a pictorial drawing of a type specimen of FIG. 2 and, when subjectedto papain digestion in the presence of cystine, this immunoglobulin iscleaved on the broken lines A--A' into two Fab fragments and one Fcfragment as shown in FIG. 2, and the Fab fragments thus obtained can beused as fragment Fab in the present invention. When said IgGl is treatedwith pepsin, it is cleaved on the broken line B--B' as shown in FIG. 2,toproduce a dimer, (F(ab')₂), of Fab' part consisting of the Fab partand the hinge part which is shaded with oblique lines in the figure. TwoFab' fragments can also be obtained by cleaving the disulfide bond inthe hinge part reductively, for instance, with the use of a thiolreagent or by cleaving it by means of sulfonation with sulfite ions.Since this Fab' fragment has an antibody activity like Fab fragment(though it does not have an ability to couple to complements), it can beused as fragment Fab of the present invention. In the present invention,so far as the fragmentFab has an antibody activity, said Fab fragment orFab' fragment may be theone chemically modified.

Also, substantial fragment Fab having at least one mercapto radical canbe converted into a fragment Fab having an active disulfide radicalwhich is expressed by the following formula (V):

    --S--Z                                                     (V)

(where Z indicates ##STR1##by allowing it to react with2,2'-dithiopyridine, 4,4'-dithiopyridine, or5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) and the thusconverted fragment Fab can be used as the fragment Fab of the presentinvention.

The thus obtained fragment Fab is used for the preparation of antitumorprotein hybrid according to the present invention just as it is so longasit has at least one thiol radical (--SH), S-sulfo radical (--S--SO₃⁻), or active disulfide radical in the fragment but in other cases it isused after it has been changed into a fragment having atleast one thiolradical, S-sulfo radical, or active disulfide radical by cleaving atleast one of the disulfide bonds in the chains (in the H chains or the Lchains) and the disulfide bonds between the chains (between the H chainsand the L chains) according to a publicly known method. The number ofthiol radicals, S-sulfo radicals, or active disulfide radicals in thefragment Fab should preferably be in the range of 1-5 (corresponding tom=1-5 in the formula (I)) and it is especially preferable to have thenumber of thiol radicals, S-sulfo radicals, or active disulfide radicalswhich are formed by cleaving the bonds between the chains within therange of 1-2 (corresponding to m=1-2 in the formula(I)).

What is called ricin in the present invention is a protein toxin whichcan be extracted and purified from the seeds of Ricinus Communisaccording to a publicly known method, for instance, a method proposed byS. Alsnes and A. Pihl in Biochemistry, vol. 12, pp. 3121-3126, 1973.Ricin consists of subunit A having a molecular weight of 32,000 andsubunit B having a molecular weight of 34,000, both of which areconnected to each other by adisulfide bond (FIG. 3, (a)). When ricin istreated with a reductant, it isdivided into subunit A and subunit B,each having at least one mercapto radical (--SH) as shown in FIG. 3,(b). Before ricin is divided apart, it has a very strong toxicityagainst animals; however, subunit B alone has only a weak toxicity andsubunit A has a weaker toxicity. It is assumed that ricin displays itscytotoxicity inhibiting the biosynthesis of protein by deactivating acomponent which is indispensable for lengtheningthe peptide chains.Subunit A has an activity to inhibit the biosynthesis of protein in thecell-free system, while subunit B does not have such activity but anability to couple to the receptor of a cell which is not seen withsubunit A. In the present invention, subunit A is used.

In the present invention, when fragment Fab of antitumor immunoglobulinhaving at least one thiol radical, S-sulfo radical, or active disulfideradical in the fragment is made to react directly with subunit A ofricin having at least one thiol radical in the fragment under thereaction conditions mentioned later, antitumor protein hybrids expressedby the undermentioned formula (II') which corresponds to theaforementioned formula (I) wherein n=0 are obtained.

    Fab--S.sub.1 --S.sub.2 --RA).sub.m                         (II')

(where the definitions of Fab, RA, m, S₁ and S₂ are the same as thosegiven in case of formula (I)). Of these, the one which has a structureexpressed by the following formula (II) is especially preferablefrom theviewpoint of ease of preparation, separation and purification:

    Fab--S.sub.1 --S.sub.2 --RA).sub.p                         (II)

(where the definitions of Fab, RA, S₁ and S₂ are same as those given incase of formula (I): p indicates 1 or 2).

In the present invention, the divalent organic radical, which isexpressed by X where n=1 in the aforementioned formula (I), means anorganic radicalarising from a cross-linking agent having in the moleculeat least two functional groups capable of forming a sulfide bond (--S--bond) by reacting with a thiol radical (--SH). Such cross-linking agentsinclude, for instance, a dimaleimide compound which is expressed by theundermentioned formula (III) and a bishalocarbonyl compound which isexpressed by the formula (IV) as the most preferable ones.##STR2##(where Y indicates a divalent organic radical). ##STR3##(where Zindicates a divalent organic radical, X₁ and X₂ are the same or differfrom each other indicating bromine or iodine).

Specific examples of dimaleimide compound expressed by theabovementioned formula (III) are, for instance,N,N'-(1,2-phenylene)dimaleimide, N,N'-(1,4-phenylene)dimaleimide,4,4'-bis(maleoylamino) azolenzene, and bis(N-maleimidomethyl)ether.Specific examples of bishalocarbonyl compoundexpressed by the formula(IV) are N,N'=alkylenebis(bromoacetamide) andN,N'-alkylenebis(iodoacetamide) (wherein the alkylene radical has 2-15carbon atoms).

The antitumor protein hybrid of the present invention can be preparedaccording to the methods given in the following.

(1) A method to make a substantial fragment Fab which has at least oneS-sulfo radical or active disulfide radical expressed by theaforementioned formula (V) in the fragment react with a subunit A whichhas at least one thiol radical in the fragment.

In this method, it is preferable to use a ratio of 0.3 to 3 moles ofsubunit A to 1 mole of fragment Fab. The reaction can be conducted bymixing fragment Fab and subunit A in a buffer whose pH is in the rangeof 5-10 to make a total protein concentration of 0.5 to 100 mg/ml (morepreferably 1 to 20 mg/ml) and leaving the mixture standing at 0° to60°C. or dialyzing the reaction mixture against the buffer having the samepH value as the reaction mixture. The reaction time generally extendsover a period of 30 minutes to 3 days, depending upon the scale andconditions of the reaction. The separation of the hybrid thus composedoffragment Fab and subunit A from the reaction mixture and thepurification can be carried out according to a usual procedure, forinstance, dialysis or column chromatography of a molecular sieve effect.

The method mentioned above allows the reaction to proceed smoothly undervery moderate conditions to produce a highly purified hybrid. The methodalso has the advantage of permitting the selective formation of hybridcomposed of fragment Fab and subunit A (as compared to the formation ofhybrid between fragments Fab themselves or between subunits A themselvescoupled by the disulfide bond).

(2) A method for binding a substantial fragment Fab which has at leastone thiol radical in the fragment and a substantial subunit A which hasat least one thiol radical in the fragment with the use of either of theaforementioned cross-linking agents expressed by the formulae (III) and(IV).

In the above method, the reaction can be conducted by bringing fragmentFab, cross-linking agent and subunit A into contact with each othersimultaneously; however, it is preferable to carry out the preparationof the hybrid by making subunit A react with the reaction productobtained byfirst allowing fragment Fab to react with the cross-linkingagent or by making fragment Fab react with the reaction product obtainedby first allowing subunit A to react with the cross-linking agent. Inthe former case, it is preferable to use 0.8 to 6 moles of thecross-linking agent and subunit A respectively to 1 mole of fragmentFab. In the latter case, it is preferable to use 0.8 to 3 moles of thecross-linking agent and 0.2 to 3 moles of fragment Fab to 1 mole ofsubunit A. The reaction is startedat 0° to 60° C. with stirring with theaddition of the cross-linking agent dissolved in a small amount ofsolvent such as N,N-dimethylformamide, dimethyl sulfoxide,1,2-dimethoxyethane, methanol, ethanol, acetone, etc. to a solution offragment Fab or subunit A bufferedat a pH of 6 to 10 (the proteinconcentration being preferably controlled to 0.5 to 100 mg/ml, or morepreferably to 1 to 20 mg/ml). After the removal of the cross-linkingagent left unreacted by means of dialysis or column chromatography of amolecular sieve effect, another component (subunit A or fragment Fab)solution buffered at a pH of 6 to 10 (the preferable ranges of proteinconcentration being the same as mentioned above) is added to carry outthe reaction at 0° to 60° C. The separation, and purification as well,of the thus obtained hybrid of fragment Fab and subunit A from thereaction mixture can be effected according to a usually adopted methodsuch as column chromatography of a molecular sieve effect.

(3) A method in which fragment Fab of the antitumor immunoglobulin whichhas at least one thiol radical in the fragment and subunit A of ricinwhich has at least one thiol radical in the subunit are subjected to theoxidative reaction in the presence of each other to have them both boundby the disulfide bond. As for the oxidative reaction, any of the airoxidation method, method of oxidation with the use of o-iodobenzoic acidand method in which oxidation is effected with o-phnanthroline andcupric sulfate may be adopted.

In the present invention, (1) and (2) of the abovementioned methods areespecially preferable.

The antitumor protein hybrid of the present invention consists of amoiety comprising subunit A which demonstrates toxicity against tumorcells and amoiety substantially comprising fragment Fab whichselectively recognizes atumor cell and works as a carrier to guide saidsubunit A to the tumor celland take subunit A into the cell as well andthis hybrid has excellent characteristics mentioned below.

(1) Since the hybrid of the present invention does not contain the Fcpart of the immunoglobulin, nonspecific binding to Fc receptors on thecell membrane with the Fc part is avoided and this fact allows theantibody activity or performance of the fragment Fab to selectivelycouple to the antibody to be demonstrated predominantly.

(2) It is known that, when a xenogeneic immunoglobulin is used, it isthe Fc part that has the strongest antigenicity. In case of the hybridaccording to the present invention, since it does not contain the Fcpart of the immunoglobulin, the antigenecity of the xenogeneicimmunoglobulin is reduced remarkably.

(3) It is known that, in case of the ricin molecule, it is the subunit Bthat has the ability to couple to the receptor of cells (normal cellsand tumor cells) and that the subunit A can be taken into the cell bymeans ofthe coupling of the subunit B to the cell membrane todemonstrate the cytotoxicity. However, since the hybrid of the presentinvention does not contain the subunit B, the hybrid of the presentinvention scarcely demonstrates cytotoxicity to normal cells.Furthermore, since it does not contain the subunit B, the antigenecityof ricin is also reduced.

(4) The hybrid of the present invention has a moiety substantiallycomprising the fragment Fab obtained from the antitumor immunoglobulinandthis moiety specifically recognizes a tumor cell and cause the tumorcell to take in specifically the moiety comprising the subunit A ofricin. The subunit A thus taken in demonstrates a remarkablecytotoxicity to the tumor cell.

The present invention is described in detail by the following examples.

Example 1 (a) Preparation of fragment Fab' of antitumor immunoglobulin

Mouse leukemia L 1210 cells transplanted successively on DBA/2 Cr miceweretaken out of the ascites of a DBA/2 Cr mouse. An emulsion preparedfrom about 10⁶ of those cells and Freund's complete adjuvant (immunoadjuvant) was intravenously injected into a rabbit. After that 10⁶ L1210 cells, together with the adjuvant, were subcutaneously injectedthreetimes at one-week intervals, and the rabbits were bled seven daysand ten days after the day of final injection. The blood thus obtainedwas mixed and serum was separated from the mixed blood and wasinactivated at 56° C. for 30 minutes. 200 ml of saturated aqueousammonium sulfatesolution was added to 200 ml of thus obtained anti-L1210 antiserum at 4° C. and the precipitate (anti-L 1210 immunoglobulin)was separated by means of centrifugation. The precipitate thus obtainedwas dissolved in 50 ml of 0.01 M phosphate buffer (pH 7.6) and wassufficiently purified by dialysis against the same buffer to obtain asolution of anti-L 1210 immunoglobulin. The solution was subjected toDEAEcellulose column chromatography (column size 3 cm×94 cm)equilibratedwith the same phosphate buffer to obtain a solutioncontaining IgG as an unadsorbed fraction. A solution of this anti-L 1210IgG (1.2 g) in 40 ml of 0.1 M acetate buffer (pH 4.5) was prepared, andto the solution 24 mg of pepsin was added to effect pepsin digestion at37° C. for about 18 hours. The digestion product was subjected toSephadex G200 column chromatography (column size 3.5 cm×140 cm) oversilane to take out protein eluted at molecular weight of about 100,000.It was confirmed thatthis was a pure fragment F(ab')₂ by means ofelectrophoresis with sodium dodecyl sulfate-polyacrylamide gel(hereinafter referred to as SDS.PAGE) (See FIG. 4, disc 1). SDS.PAGE wascarried out according to a method proposed by K. Weber and Mr. Osborn inthe Journal of Biological Chemistry, vol. 244, pp. 4406-4412, 1969.However, the present electrophoresis was conducted with 6 M ureacontained in the solution togethe with 0.1% sodium dodecyl sulfate withthe gel concentration kept at 6%. 0.02 ml of 150 m M 2-mercaptoethanolwas added to 2.0 ml of 0.01 M tris.HCl-0.14 M sodium chloride-2 Methylenediaminetetraacetic acid (pH 8.3) containing 18.4 g of thusobtained fragment F(ab')₂ to effect reduction at 37° C. for one hour.The reduction product was subjected to Sephadex G25 columnchromatography (1.0 cm×20 cm) equilibrated with 5 m M acetatebuffer-0.14 M sodium chloride-1 m M ethylenediaminetetraacetic acid (pH5.5) to remove 2-mercaptoethanol to give fragment Fab' having one thiolradical (See FIG. 4, disc 2).

(b) Preparation of fragment Fab' having a 3-carboxy-4-nitrophenylthioradical

After F(ab')₂ was reduced exactly according to the method mentionedabove, 0.2 ml of an ethanol solution containing 50 m M5,5'-dithiobis(2-nitrobenzoic acid) was added to 2.0 ml of thus obtainedprotein solution to conduct the reaction at room temperature for 50minutes. This reaction mixture was subjected to Sephadex columnchromatography referred to in the preceeding (a) for the purpose ofremoving substances of low molecular weight to obtain Fab' which has a3-carboxy-4-nitrophenylthio radical.

(c) Preparation of ricin subunit A

Extraction and purification of ricin from the seeds of Ricinus Communisandseparation of subunit A from ricin were effected according to themethod ofS. Olsnes and A. Pihl (Biochemistry, vol. 12, pp. 3121-3126,1973). Since the obtained subunit A solution contained2-mercaptoethanol, the solution was, immediately before its use,subjected to Sephadex G25 column chromatography described in theforegoing (a) to remove 2-mercaptoethanol.Neither subunit B nor intactricin was detected in this subunit A solution on its SDS.PAGE (See FIG.4, disc 3).

(d) Preparation of antitumor protein hybrid

1.8 ml of 5 m M acetate buffer-0.14 M sodium chloride-1 m M ethylenediaminetetraacetic acid (pH 5.5) containing 13.3 mg of Fab' having anactive disulfide radical obtained in the preceding (b), 1.8 ml of 5 m Macetate buffer-0.14 M sodium chloride-1 m M ethylenediaminetetraaceticacid (pH 5.5) containing 6.5 mg of subunit A obtained in the preceding(b), and 0.3 ml of 0.4 M phosphate buffer-0.01 Methylenediaminetetraacetic acid (pH 7.23) were mixed together andallowed to stand at room temperature for 4.5 hours. After the additionof 5 mg of iodoacetamide, the mixture was left standing at roomtemperature for 15 minutes and then subjected to sephadex G150(superfine) column chromatography (1.4 cm×90 cm) equilibrated withsaline. 2.1-ml fractions were collected and the absorbances at 280 mμ ofthe respective fractions were measured, the results of whichmeasurements are shown in FIG. 5. There are three peaks in the figureand they are named peaks I, II, and III from the left, whose proteinidentification was made by means of SDS-PAGE. Peak II is fragment Fab'as per disc 7 and peak III is subunit A as per disc 8. Peak I shows oneband as in disc 6 at a position near to that of F(ab')₂ or dimer ofsubunit A. However, wheneither peak I and F(ab')₂ or peak I and dimer ofsubunit A are mixed and subjected to electrophoresis, the three kinds ofproteins show themselves clearly differing from each other as per discs9 and 10. Furthermore, when peak I is reduced moderately with2-mercaptoethanol, it divides into Fab' and subunit A as per disc 11.From the above, it has been confirmed that peak I is a hybrid comprisingFab' and subunit A coupled together by a disulfide bond at the ratio of1:1.

(e) Cytotoxicity of antitumor protein hybrid

Fractions falling under the shaded part of FIG. 5 were collected toobtain saline containing the protein hybrid (1.1 mg/ml) of the presentinvention.This aqueous solution was used for measuring cytotoxicity ofthe protein hybrid of the present invention against mouse lukemia L 1210cells.

In the culture wells having a bottom area of 2.0 cm², 1.4 ml of themedium RPMI 1640 (containing 10% fetal calf serum and 20 μM2-mercaptoethanol), 0.1 ml of L 1210 cell suspension (5×10⁵ cells/ml),and 0.1 ml of the test sample (ricin or protein hybrid of the presentinvention) were mixed, and furthermore, as occasion may require,anti-rabbit Fab' antibody (130 μg/ml) (which antibody is obtained byimmunizing a goat with rabbit Fab') or 0.1 mol (final concentration) ofα-lactose may be added as a toxicity inhibitor. The culture was carriedout a 37° C. in an atmosphere of 5% CO₂ for 42 hours. Thereafter, thecytotoxicity was observed under a phase-contrast microscope. When all ofthe globular cells were found damaged, the result is indicated by +++;when 90-50% were found damaged, the result is indicated by ++; when50-10% were found damaged, the result is indicated by +; and when theresult was the same as that of the control, it is indicated by -, Theresults are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cytotoxicity of antitumor                                                     protein hybrid against                                                        L 1210 cells                                                                                       Protein hybrid                                                                of the present                                                    (Ricin (ng/ml)                                                                            invention (ng/ml)                                        Inhibitor  70      7       0.7 7000   700  70                                 ______________________________________                                        None       +++     ++      +   ++     +    -                                  Anti-rabbit                                                                              +++     ++      +   -      -    -                                  Fab' antibody                                                                 α-lactose                                                                          ++      -       -   ++     +    -                                  Anti-rabbit Fab'                                                                         ++      -       -   -      -    -                                  antibody +                                                                    α-lactose                                                               ______________________________________                                    

As Table 1 shows clearly, ricin itself has nonselective strong toxicityandthis toxicity is not influenced by anti-rabbit Fab' antibody butreduced by α-lactose which is a substance to inhibit the binding ofricin and cells.

On the other hand, though the protein hybrid of the present inventionhas atoxicity weaker than ricin, it still has enough cytotoxicity. Andfrom the fact that this toxicity can be completely suppressed by theanti-rabbit Fab' antibody, it is known that the moiety comprisingfragment Fab' of theprotein hybrid plays an important role in thiscytotoxicity. It is also made known that, since this toxicity, differentfrom ricin, is quite free from the influence of α-lactose, the toxicitydoes not arise from any contamination with ricin and that the toxicityis displayed by a mechanism different from that of ricin. From thesefacts, the protein hybrid of the present invention is expected todisplay specific toxicity against tumor cells which the fragment Fab'can recognize.

Example 2 (a) Preparation of fragment Fab' having 4-pyridylthio radical

0.2 ml of an ethanol solution containing 50 m M 4,4'-dipyridyldisulfidewasadded to 2.0 ml of the protein solution after the reduction ofF(ab')₂conducted according to Example 1, (a), and the mixture wasallowed to reactat a room temperature for 30 minutes. This reactionsolution was subjected to Sephadex G25 column chromatography to removesubstances of low molecular weight, thus obtaining Fab' having a4-pyridylthio radical.

(b) Preparation of antitumor protein hybrid

12.5 mg of fragment Fab' having an active disulfide radical obtained inthepreceding (a) and 6.1 mg of subunit A of ricin obtained in Example 1,(c), were mixed in 1.57 ml of 0.1 M phosphate buffer-2 m Methylenediaminetetraacetic acid (pH 6.6) to carry out the couplingreaction at room temperature for 3 hours. After that, the sameprocedures as taken in Example 1 were followed to obtain the proteinhybrid, the object of the present invention, comprising fragment Fab'and subunit A linked by a disulfide bond.

Example 3

13.1 mg of fragment Fab' having a thiol radical obtained in Example 1,(a),and 5.8 mg of subunit A of ricin obtained in Example 1, (c), weremixed in 3.6 ml of 0.1 M glycine buffer-2 m M ethylenediaminetetraaceticacid (pH 9.15) to carry out the coupling reaction at room temperaturefor 8 hours. Thereafter, the same procedures as taken in Example 1 werefollowed to obtain the protein hybrid, the object of the presentinvention, comprisingfragment Fab' and subunit A linked by a disulfidebond.

Example 4 (a) Preparation of fragment Fab' having an S-sulfo radical

1.7 mg of sodium sulfite and 0.7 mg of sodium tetrathionate were addedto 2.0 ml of 0.1 M Tris.HCl-2 m M ethylenediaminetetraacetic acid (pH7.8) containing 18.3 mg of fragment F(ab')₂ obtained in Example 1, (a),tocarry out the sulfonation reaction at 37° C. for one hour. Thesulfonated solution was subjected to Sephadex G25 column chromatographyaccording to Example 1, (a), to obtain fragment Fab' having an S-sulforadical.

(b) Preparation of antitumor protein hybrid

130 mg of fragment Fab' having an S-sulfo radical obtained in theabovementioned (a) and 5.6 mg of subunit A of ricin obtained in Example1,(c), were mixed in 1.82 ml of 5 m M acetate buffer-0.14 M sodiumchloride-1m M ethylenediaminetetraacetic acid (pH 5.5). The admixturewas dialyzed at4° C. for three days against 1 l of 0.1 M glycinebuffer-2 m M ethylenediamine tetraacetic acid (pH 9.15) to effect thereaction. Thereafter, the same procedures as taken in Example 1 werefollowed to obtain the protein hybrid, the object of the presentinvention, comprisingfragment Fab' and subunit A linked each other by adisulfide bond.

Example 5

A fragment Fab' solution (7 mg/ml) was prepared by dissolving fragmentFab'of anti-L 1210 immunoglobulin IgG having one thiol radical obtainedin Example 1, (a), in a mixed solution consisting of 2 parts by volumeof 0.1M sodium phosphate buffer (pH 6.0) and 1 part by volume ofacetone. A suspension of a cross-linking agent,N,N'-(1,4-phenylene)dimaleimide (PDM)in acetone (5 mg/ml) was separatelyprepared.

0.1 ml of the PDM solution was added dropwise to 1.0 ml of the fragmentFab' solution and the reaction was allowed to proceed at roomtemperature for 30 minutes. Acetone was removed from the obtainedreaction mixture with the use of an evaporator and further insolublesubstances were removed by means of centrifugation conducted at 10,000rpm for 30 minutes.The solution thus obtained was subjected to SephadexG25 column chromatography equilibrated with 0.1 M sodium phosphatebuffer (pH 7.0) togive a solution of fragment Fab' having a PDM residue.

The thus obtained solution of fragment Fab' having a PDM residue wasadmixed with subunit A of ricin having one thiol radical preparedaccording to Example 1, (c), in such a way as to have the fragmentFab'-subunit A molar ratio of 1:0.7 and the coupling reaction wasallowed to proceed at 4° C. for 24 hours and further at 37° C. for onehour. The obtained reaction solution was subjected to Sephadex G150column chromatography under the same conditions as Example 1. As aresult of measurement of the absorbance at 280 mμ of the fractions,three peaks were observed as in the case of Example 1. The 37th and 38thfractions which came under peak I showed the precipitation reaction withgoat anti-rabbit IgG anti-serum and also with guinea pig anti-ricin Aantiserum. It was confirmed that their molecular weight was 76,000 onelectrophoresis conducted with sodium dodecyl sulfate and also that theycould not be divided into fragment Fab' and subunit A when treated with2-mercaptoethanol. From these facts it was confirmed that the 37th and38th fractions contained the protein hybrid aimed at for the object ofthepresent invention, or more particularly, that these fractionscontained theprotein hybrid in which fragment Fab' and subunit A werelinked together byPDM, a cross-linking agent, via the respective sulfuratoms. It was also found that this protein hybrid had cytotoxicityagainst L 1210 almost equal to potency to the one obtained in Example 1.

Example 6

According to Example 5, antitumor protein hybrid was obtained, in whichfragment Fab' and subunit A were linked to each other by a cross-linkingagent of N,N'-(1,2-phenylene)dimaleimide via the respective sulfuratoms, wherein N,N'-(1,3-phenylene)dimaleimide was used in the place ofPDM whichwas used in Example 5.

Example 7

According to Example 5, antitumor protein hybrid was obtained, in whichfragment Fab' and subunit A were linked to each other by a cross-linkingagent of 4,4'-bis(maleoylamine)azobenzene via the respective sulfuratoms,wherein 4,4'-bis(maleoylamino)azobenzene was used in the place ofPDM whichwas used in Example 5.

Example 8

Fragment Fab' of anti-L 1210 immunoglobulin IgG having one thiol radicalobtained according to Example 1, (a), was dissolved in a mixed solutionconsisting of 3 parts by volume of 0.1 M sodium phosphate buffer (pH7.5) and 1 part by volume of N,N-dimethylformamide at the concentrationof 7 mg/ml to prepare a solution of fragment Fab'. Besides thissolution, a solution was prepared by dissolving a cross-linking agent ofN,N'-ethylenebis(iodoacetamide) in N,N-dimethylformamide at aconcentration of 6 mg/ml.

0.1 ml of the N,N'-ethylenebis(iodoacetamide) solution was addeddropwise to 1.0 ml of the fragment Fab' solution, and the reaction wasallowed to proceed at room temperature for one hour. The obtainedreaction mixture was purified by column chromatography on Sephadex G25equilibrated with 0.1 M sodium phosphate buffer (pH 7.5) to give asolution of fragment Fab'having N,N'-ethylenebis(iodoacetamide) residue.

The subunit A of ricin having one thiol radical prepared according toExample 1, (c), was added to the solution of fragment Fab' havingN,N'-ethylenebis(iodoacetamide) residue obtained as mentioned above tomake the molar ratio of fragment Fab' havingN,N'-ethylenebis(iodoacetamide) residue to subunit A 1:0.5 and mixed.After that, the procedures were followed as in the case of Example 5 togive the protein hybrid of the present invention in which fragment Fab'and subunit A were linked to each other by a cross-linking agent ofN,N'-ethylenebis(iodoacetamide) via the respective sulfur atoms. Thisprotein hybrid had a remarkable cytotoxicity against L 1210 cells almostequal in potency to the one obtained according to Example 1.

Example 9

According to Example 8, antitumor protein hybrid was obtained, in whichfragment Fab' and subunit A were linked to each other by a cross-linkingagent of N,N'-hexamethylenebis(iodoacetamide) through the medium of therespective sulfur atoms, wherein N,N'-hexamethylenebis(iodoacetamide)was used in the place of N,N'-ethylenebis(iodoacetamide) which was usedin Example 8.

Example 10

According to Example 8, antitumor protein hybrid was obtained, in whichfragment Fab' and subunit A were linked to each other by a cross-linkingagent of N,N'-undecamethylenebis(iodoacetamide) through the medium ofthe respective sulfur atoms, whereinN,N'-undecamethylenebis(iodoacetamide) was used in the place ofN,N'-ethylenebis(iodoacetamide) which was used inExample 8.

Example 11

According to Example 5, antitumor protein hybrid was obtained, in whichfragment Fab' and subunit A were linked to each other by a cross-linkingagent of bis(N-maleimidemethyl)ether through the medium of therespective sulfur atoms, wherein bis(N-maleimidemethyl)ether was used inthe place ofPDM which was used in Example 5.

What is claimed is:
 1. A process for preparing an antitumor proteinhybrid which is expressed by the following formula (II'), which processcomprises reacting the substantial fragment Fab of an antitumorimmunogloblin which has at least one S-sulfo radical or an activedisulfide radical expressed by the following formula (V) with thesubunit A of ricin which has at least one thiol radical in the fragment:

    --S--Z                                                     (V)

where Z indicates ##STR4## wherein Fab indicates a moiety which issubstantially the fragment Fab of an antitumor immunoglobulin; RAindicates a moiety which is the subunit A of ricin; S₁ and S₂ are bothsulfur atoms, S₁ indicating a sulfur atom arising fron the disulfidebond in the immunoglobulin and S₂ a sulfur atom arising from thedisulfide bond in ricin; and m represents an integer of 1 to
 5. 2. Aprocess for preparing an antitumor protein hybrid which is expressed bythe following formula (I'), which process comprises binding thesubstantial fragment Fab of an antitumor immunoglobulin which has atleast one thiol radical in the fragment with the subunit A of ricinwhich has at least one thiol radical in the fragment with the use of acompound which has at least two functional groups capable of reactingwith a thiol radical

    Fab--S.sub.1 --X--S.sub.2 --RA).sub.m                      (I')

wherein Fab indicates a moiety which is substantially the fragment Fabof an antitumor immunoglobulin; RA indicates a moiety which is thesubunit A of ricin; X indicates a divalent organic radical; S₁ and S₂are both sulfur atoms, S₁ indicating a sulfur atom arising from thedisulfide bond in the immunoglobulin and S₂ a sulfur atom arising fromthe sulfide bond in ricin; and m represents a integer of 1 to
 5. 3. Theprocess of claim 1, wherein said substantial fragment Fab of anantitumor immunogloblin is the substantial fragment Fab of an antitumorimmunogloblin having at least one S-sulfo radical.